TRPC6 is a mechanosensitive channel essential for ultrasound neuromodulation in the mammalian brain
Yumi Matsushita, Kaede Yoshida, Miyuki Yoshiya, Takahiro Shimizu, Satoshi Tsukamoto, Nobuki Kudo, Yuichi Takeuchi, Makoto Higuchi, Masafumi Shimojo
Proceedings of the National Academy of Sciences 2024, 121 · 10.1073/pnas.2404877121
Abstract
Ultrasound neuromodulation has become an innovative technology that enables noninvasive intervention in mammalian brain circuits with high spatiotemporal precision. Despite the expanding utility of ultrasound neuromodulation in the neuroscience research field and clinical applications, the molecular and cellular mechanisms by which ultrasound impacts neural activity in the brain are still largely unknown. Here, we report that transient receptor potential canonical 6 (TRPC6), a mechanosensitive nonselective cation channel, is essential for ultrasound neuromodulation of mammalian neurons in vitro and in vivo. We first demonstrated that ultrasound irradiation elicited rapid and robust Ca 2+ transients mediated via extracellular Ca 2+ influx in cultured mouse cortical and hippocampal neurons. Ultrasound-induced neuronal responses were massively diminished by blocking either the generation of action potential or synaptic transmission. Importantly, both pharmacological inhibition and genetic deficiency of TRPC6 almost completely abolished neuronal responses to ultrasound. Furthermore, we found that intracerebroventricular administration of a TRPC6 blocker significantly attenuated the number of neuronal firings in the cerebral cortex evoked by transcranial ultrasound irradiation in mice. Our findings indicate that TRPC6 is an indispensable molecule of ultrasound neuromodulation in intact mammalian brains, providing fundamental understanding of biophysical molecular mechanisms of ultrasound neuromodulation as well as insight into its future feasibility in neuroscience and translational research in humans.
Abstract via europepmc.
Exposures
Exposure 1: Cultured cortical/hippocampal neurons (in vitro Ca2+ imaging)
Target: cultured neurons — “cultured mouse cortical and hippocampal neurons”
Device: other named manufacturer · NEPA GENE · ST-TM1-6 transducer with SonoPore KTAC-4000 amplifier ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | 5 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 100 | ✓✓✓ |
| Duty cycle (%) | 50pulse duration × PRF gives 50% | ✓✓✓ |
| Sonication duration (s) | 0.1, 0.2, 0.5swept | ✓? |
| Free-field pressure (kPa) | not reported | |
|---|---|---|
| Free-field Isppa (W/cm²) | 0.38 | ✓✓✓ |
| Free-field Ispta (W/cm²) | 0.017, 0.19, 0.294swept | ✓?⚑ |
| In-situ estimate | not applicable | |
| In-situ pressure (kPa) | not applicable | |
| In-situ Isppa (W/cm²) | not applicable | |
| In-situ Ispta (W/cm²) | not applicable |
Neurons were stimulated with ultrasound waves at a frequency of 1 MHz. Each stimulus consisted of 100 Hz PRF with a duty cycle of 50%. Neurons were sonicated at different stimulation parameters in terms of irradiation intensities (17, 190, 294 mW/cm2) and durations (100, 200, 500 msec); an intensity of 190 mW/cm2 and a duration of 500 msec were chosen for all subsequent experiments.
Exposure 2: Transcranial ultrasound to cerebral cortex (in vivo electrophysiology)
Target: cerebral cortex — “cerebral cortex (2.06 mm posterior, 0.36 mm rightward from bregma)”
Device: other named manufacturer · NEPA GENE · ST-T1-3 transducer with SonoPore KTAC-4000 amplifier ✓
| Waveform | pulsed | |
|---|---|---|
| Fundamental frequency (kHz) | 1,000 | ✓✓✓ |
| Pulse duration (ms) | 5 | ✓✓✓ |
| Pulse repetition frequency (Hz) | 100 | ✓✓✓ |
| Duty cycle (%) | 50pulse duration × PRF gives 50% | ✓✓✓ |
| Sonication duration (s) | 0.1 | ✓✓✓ |
| Free-field pressure (kPa) | 160 | ✓✓✓ |
|---|---|---|
| Free-field Isppa (W/cm²) | not reported | |
| Free-field Ispta (W/cm²) | 0.07, 0.13, 0.21, 0.35, 0.52, 0.73swept | ✓✓✓ |
| In-situ estimate | not reported | |
| In-situ pressure (kPa) | not reported | |
| In-situ Isppa (W/cm²) | not reported | |
| In-situ Ispta (W/cm²) | not reported |
Transcranial ultrasound irradiation of 1 MHz in 100 Hz PRF and 50% duty cycle, the same parameter as in our in vitro experiments. Basic parameters of the ultrasound burst pulse sequence are the same as that used for in vitro studies, but the train contains 10 burst pulses, and the duration is 100 msec. At each recording site, several recording sessions at different ultrasound intensities (0, 70, 130, 210, 350, 520, 730 mW/cm2) were performed; ultrasound stimulations were performed 20 times with a random interstimulus interval ranging from 10 to 20 s.
Flags from extraction
model_system— Study combines an in vitro cultured-neuron mechanism experiment with an in vivo transcranial mouse experiment; n_subjects and subject_unit reflect only the in vivo animal experiment (3 mice).n_sessions_per_subject— Paper reports 73 pre- and 80 post-drug recording sessions pooled across three mice, without stating sessions per individual mouse.exposures[0].free_field.ispta_w_cm2— Three intensities (17, 190, 294 mW/cm2) were tested as a sweep; only the 190 mW/cm2 condition was independently confirmed to match the calculated ISPTA (0.19 W/cm2).sham_type— The 0 mW/cm2 condition in the in vivo intensity sweep is treated as the inactive/no-output control condition; no separate device-off sham session is described.